Ecological preferences of freshwater Ulva flexuosa (Ulvales; Ulvophyceae): development of macroalgal mats in a Tulce fishpond (Wielkopolska Region, Poland)

The Ulva (Chlorophyta) genus belongs to the cosmopolitan group of macroalgae (Hayden et al. 2003) that grow primarily in seawater and estuaries (McAvoy & Klug 2005). With a very high rate of biomass increase in eutrophic waters, these algae form “green tides”, which have been repeatedly found in European and Asian coastal waters (Blomster et al. 2002; Liu et al. 2010; Pang et al. 2010). Moreover, Ulva thalli have been observed at inland sites in Europe. In the 19th century, Ulva thalli were recorded in inland salt pans and waters located near mineral water source areas (Göppert & Cohn 1850; Hansgirg 1892). At several sites, the waters of which contained high levels of mineral compounds, Ulva has been recognized as one of the native elements of the halophilic flora in these areas (or flora marina in aquatic ecosystems of inland areas not affected by the Baltic Sea water). In the 20th century, Ulva populations were also recorded in freshwater ecosystems, such as lakes and lowland rivers and ponds, where the NaCl level did not exceed 20 mg l^-1 (Messyasz & Rybak 2009, 2011; Rybak et al. 2014). The abundant occurrence of Ulva has been observed in European lakes, ponds and rivers with optimal conditions for its development, becoming an obvious dominant on the water surface (Kaštovský et al. 2011). The occurrence of ten Ulva species was reported in the Baltic waters along the littoral sites (Pliński & Jóźwiak 2004). In the inland ecosystems of Europe, which are not exposed to the impact of sea and ocean waters, the Ulva species have been recorded at more than 220 sites (Rybak et al. 2014; Rybak 2015).

Species of the Ulva genus demonstrate considerable morphological plasticity, the expression of which is influenced by environmental conditions, such as nutrient concentration and water salinity. Populations of a given species may produce either tube-like or leaf-like thalli at the bottom or top part of the thallus, which complicates the identification of taxa. It has been reported that a wide morphological plasticity is strongly expressed in the case of fresh water Ulva populations, the thalli of which grow to sizes not observed in bibliographical references (Messyasz & Rybak 2011). Currently, phylogenetic methods have become increasingly popular for the identification of Ulva species. Among the markers used, ITS1 – 5.8S rRNA – ITS2, nrDNA, rbc L are the most common; additionally, a special marker is used for SSU rRNA to create more informative sequence alignments and cladograms with better topological support (Mareš et al. 2011; Melton et al. 2015).

However, the research on freshwater populations of Ulva are focused on several aspects. The available studies focus on (i) the analysis of phylogenetic affinity with sea populations (Mareš et al. 2011; Chen 2015), (ii) the expression of ULL (Ulva-Lectin-Like) genes, which are most likely responsible for Ulva resistance to the conditions of gradual mineralization of waters (Ichihara et al. 2011, 2013), and (iii) the bioaccumulation of heavy and light metals (Rybak et al. 2011; Rybak et al. 2012a, b; Rybak et al. 2013). On the other hand, there are no studies concerning the monitoring of the freshwater Ulva population development at inland sites, and such studies are essential given the increasingly frequent occurrence of macroalgae mats with Ulva flexuosa in freshwater ecosystems of Europe.

This objective of this study is to provide additional information for the previous ecological studies of freshwater Ulva populations in inland water ecosystems. Indeed, detailed ecological analyses of parameters that influence the occurrence of Ulva species in freshwaters are needed. Therefore, the objective of this work was to determine the influence of physio-chemical factors on the colonization and the growth of limnic Ulva populations in freshwater ecosystems using pond samples. An additional concern was to evaluate the impact of Ulva thalli on local disturbances in the concentration of nutrients.

The abundant occurrence of Ulva flexuosa in freshwater ecosystems in the form of dense mats which cover considerable areas of water surface is not a common phenomenon. In Europe, several to dozens freshwater populations of U. flexuosa subsp. pilifera were observed creeping along the basin floor or floating freely on the water surface (Messyasz & Rybak 2009; Mareš et al. 2011). In 2007-2008, the freshwater population of U. flexuosa was observed forming the mats of just 4 m² (Messyasz & Rybak 2011). In that case, high levels of ammonia nitrogen and the increasing water depth were the main factors determining the process of mat formation. Under such conditions, the records evidenced the highest concentration of thalli in 1 dm³ of water (Messyasz & Rybak 2009; Messyasz & Rybak 2011). In the studies of the freshwater population of U. flexuosa originating from the fish pond, the water depth and concentration of ammonia nitrogen were not as important as the level of conductivity and chloride concentration. These two parameters determined the chemical specificity of the studied stand. García & Aboal (2014) have also indicated the conductivity as the main determinant of U. flexuosa occurrence in their field research on the dynamics of the macroalgae population development in the area of the Mediterranean marshes. However, in the fish pond where the U. flexuosa “green tide” was monitored, the concentration of nutrients, and in particular ammonia (> 4 mg l^-1), was high.

The abundant occurrence of Ulva in saltwater ecosystems is connected with higher concentrations of nutrients in water, specifically nitrogen and phosphorus (Valiela et al. 1997). Ulva species which form the “green tide” are mainly: U. intestinalis, U. lactuca (Blomster et al. 2002), U. ohnoi (Hiraoka et al. 2003), U. pertusa (Ishii et al. 2001), and U. prolifera (Liu et al. 2010). The correlation between quick biomass growth of Ulva species thalli and high concentrations of nutrients in water often made thalli of such species as U. rotundata, U. reticulata, and U. lactuca useful as nutrient biological filters, e.g. in breeding ponds with shrimps and saltwater fish (Neori et al. 1991; Jimenez del Rio et al. 1996; Hernández et al. 1997; Schroeder et al. 2013). Some species like U. lactuca or U. intestinalis are used for monitoring of over-fertilized waters (Reed & Moffat 2003). For this reason, Ulva opportunistic macroalgae have been included on the list of water quality indicators, which was developed for the purposes of the European Water Framework Directive (WFD 2000).

Brault & Quéquiner (1989) demonstrated that Ulva gigantea biomass would keep growing under the influence of increasing concentration of ammonia nitrogen. In the laboratory studies of the freshwater population of U. flexuosa (Rybak et al. 2012c), the contribution of organic nitrogen was measured at the level of 1.79-2.29% dry mass (KNO₃ being the source of nitrogen). On the other hand, the thalli incubated on NH₄Cl culture medium featured the concentration of organic nitrogen from 1.89 to 2.48% dry mass. The nitrogen level in U. flexuosa thalli was very similar to the content of nitrogen in a saltwater tubular thallus (the assay up to Enteromorpha sp. genus only) where the value of organic nitrogen in the thallus amounted to 0.7 through 3.5% dry mass (Fong et al. 1994). In their studies of U. clathrata, Copertino et al. (2009) demonstrated the highest accumulation of nitrogen in the ammonium form compared with other available nitrogen forms. Furthermore, these studies proved the lowest possible efficiency of phosphate biofiltration from the medium. A similar relationship was found by Taboada (2009) in his studies using U. reticulata and U. lactuca. In this case, the uptake of nutrients (in the form of NH₄⁺ and PO₄³) was consistent with Michaelis-Menten kinetics, which allows to conclude that the rate of nutrients’ consumption from the medium depends on the concentration of such elements in the water, but it will grow up to a certain level of nutrient concentration and a certain value of macroalga biomass. The efficiency of consumption of nitrogen in NH₄⁺ from the water contaminated by U. reticulata and U. lactuca thalli amounted to 95.8% and 93.5%, respectively. For the purpose of comparison, the efficiency of consumption of phosphorus in the PO₄^3- form by the two species was 78.6% and 78.3%, respectively (Taboada 2009). In the case of the marine species U. rotundata, the rate of ammonia nitrogen consumption reaches the level of 98% in just 30 minutes of incubation. On the other hand, the nitrate removal efficiency was at the level of 84% in the same period of incubation (Anibal et al. 2013). Although the rate of ammonia nitrogen consumption is definitely higher than in the case of nitrates following the beginning of incubation, the levels of the two nutrients in the medium will drop to zero in one hour (Anibal et al. 2013).

Furthermore, under ex situ conditions, the rates of nutrients’ consumption by the species with tubular thalli (such as U. prolifera ) were much higher than in species with leaf-shaped thalli (e.g. U. rigida ) (Kim et al. 2007). This phenomenon may be explained by the fact that the area of cell-water contact is larger in tubular thalli compared with leaf-shaped thalli (Luo et al. 2012). Many field studies have indicated that the growth of biomass in Ulva marine species from coastal waters ranges from 4 to 33 g m^-2 d^-1 (Sfriso et al. 1987, 1988; Hernández et al. 1997). The growth of Ulva biomass in basins designed for saltwater pisciculture was on average 14 gm^-2d^-1 (Israel et al. 1995).

Advanced numerical modelling of “green tides” of macroalgae demonstrated that the main drivers in the formation of large Ulva mats in the spring season at the coasts of France were high concentrations of nitrates (Perrot et al. 2014). Hence, the only method which may reduce the frequent blooming of macroalgae in coastal waters consists in the reduction of water discharge contaminated with nutrients (Beaujouan et al. 2002; Ruiz et al. 2002; Guidone & Thornber 2013). The occurrence of considerable Ulva biomass in coastal waters and on the beaches may considerably reduce the tourist attractiveness of such regions as well as produce the potential risk to human health because of the gases generated by putrefaction of thalli heaps (CEVA 2011). On the coasts of France, a horse and several dozens of wild boars died from intoxication with hydrogen sulfide which was released from under the decaying Ulva mats (Smetacek & Zingone 2013; Charlier et al. 2008). Green tides of U. flexuosa are more and more frequently observed in European lakes and rivers (Rybak et al. 2014). They may become potentially hazardous to native aquatic life forms as well as generate many problems for holiday and health resorts.

The use of Ulva marine species as biological filters (macroalgal “nutrient traps”) in fish and shrimp breeding ponds is limited to brackish or salty basins only, i.e. the sites located directly on the sea or ocean coasts. For the purpose of saltwater fish breeding (e.g. gilt-head (sea) bream – Sparus aurata ), the optimum salinity for many Ulva species (e.g. U. lactuca, U. intestinalis or U. compressa ) ranges from 15 to 37 PSU (de Paula Silva et al. 2008). Thus, Ulva marine species cannot be used as biological filters of nutrients in ponds designed for freshwater pisciculture, e.g. carp breeding. In this case, one of the alternative solutions is the use of U. flexuosa freshwater population thalli since U. flexuosa occurs in waters with salinity of up to <0.5 PSU, and the thalli will tolerate the increased salinity of up to 6 PSU (Rybak & Messyasz 2011; Rybak et al. 2014). Another thalli may come from U. limnetica, which features the largest spectrum of tolerance towards the salinity (0-30 PSU) among the identified freshwater Ulva taxa (Ichihara et al. 2013).

In the case of limnetic U. flexuosa from the studied fish pond, statistically significant differences in nitrate concentrations directly under and outside the macroalga mats were determined − up to 0.1 mg l^-1. Nevertheless, the water flow system in this fish pond features the permanent renewal and the measurements produce considerably high values.

The study presents the results of macroalgal monitoring connected with the formation of the Ulva flexuosa “green tide” in the freshwater ecosystem. The case was the first bibliographical reference describing the example of “green tides” with Ulva flexuosa formation outside the coastal ecosystems. In connection with a real risk of occurrence of “green tides” in lakes and ponds (also those used in the economy), which feature high nutrient values, a detailed analysis will be needed to study the abundant occurrence of freshwater Ulva, also on a smaller scale. Furthermore, such studies should account for the impact of Ulva blooming on aquatic biocoenosis (phytoplankton or zooplankton), since the effects of long-term impact of Ulva-containing macroalgal mats on the native freshwater hydrocenosis, e.g. lakes, remain unknown. These issues should be addressed by more detailed hydrobiological studies scheduled for many independent study sites and aquatic ecosystem types featuring various types of development.